Coarse-grained molecular dynamics simulations are a useful tool to determine conformational ensembles of proteins. Here, we show that the coarse-grained force field Martini 3 underestimates the global dimensions of intrinsically disordered proteins (IDPs) and multidomain proteins when compared with small-angle X-ray scattering (SAXS) data and that increasing the strength of protein−water interactions favors more expanded conformations. We find that increasing the strength of interactions between protein and water by ca. 10% results in improved agreement with the SAXS data for IDPs and multidomain proteins. We also show that this correction results in a more accurate description of self-association of IDPs and folded proteins and better agreement with paramagnetic relaxation enhancement data for most IDPs. While simulations with this revised force field still show deviations to experiments for some systems, our results suggest that it is overall a substantial improvement for coarsegrained simulations of soluble proteins.

Liquid–liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (i) electrostatically-mediated interactions that compact hnRNPA1 and contribute to phase separation and (ii) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation.

Intrinsically disordered proteins (IDPs) and multidomain proteins with flexible linkers show a high level of structural heterogeneity and are best described by ensembles consisting of multiple conformations with associated thermodynamic weights. Determining conformational ensembles usually involves the integration of biophysical experiments and computational models. In this review, we discuss current approaches to determine conformational ensembles of IDPs and multidomain proteins, including the choice of biophysical experiments, computational models used to sample protein conformations, models to calculate experimental observables from protein structure, and methods to refine ensembles against experimental data. We also provide examples of recent applications of integrative conformational ensemble determination to study IDPs and multidomain proteins and suggest future directions for research in the field.

Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin-2 (IL-2), a cytokine critical to T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation, and antitumor immunity by CD8 + T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Rα with higher affinity, triggers STAT5 activation, and drives CD8 + T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Last, we show that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues.

Coarse-grained molecular dynamics simulations are a useful tool to determine conformational ensembles of intrinsically disordered proteins (IDPs). Here, we show that the coarse-grained force field Martini~3 underestimates the global dimensions of IDPs when compared with small angle X-ray scattering (SAXS) data. Increasing the strength of protein-water interactions favors more expanded conformations, improving agreement with SAXS data and alleviating problems with overestimated IDP-IDP interactions.

Liquid-liquid phase separation has emerged as a new paradigm for the compartmentalization of cells without membranes. Intrinsically disordered low-complexity domains (LCDs) are often sufficient for mediating phase separation, but how their phase behavior is modulated by the presence of attached folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1, which localizes to stress granules under conditions of stress. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, the stimulusresponsive phase behavior of a number of domain deletion constructs suggests that the folded RRM domains contribute to phase separation, even in the absence of RNA. Small-angle X-ray scattering experiments show that hnRNPA1 adopts compact conformations at low ionic strength, that the protein expands considerably with increasing ionic strength, and that it reaches a maximal plateau at 300 mM NaCl. These data point to electrostatically mediated interactions that compact hnRNPA1. NMR experiments and coarse-grained MD simulations showed that the LCD interacts transiently with the RRMs, providing a mechanistic explanation for the observed stimulus-responsiveness of phase separation. Disordered and folded domains that have evolved together likely encode a multitude of interactions that can modify and regulate phase separation. Graphical AbstracthnRNPA1 phase separation is highly salt sensitive. LCD-LCD interactions can mediate phase separation over a wide range of NaCl concentrations. At low NaCl concentrations, electrostatic RRM-LCD interactions occur and can contribute positively to phase separation, but they are screened at high NaCl concentrations. The folded domains solubilize hnRNPA1 under these conditions and prevent phase separation.

Speckle-type POZ protein (SPOP) is a substrate adaptor in the ubiquitin proteasome system, and plays important roles in cell-cycle control, development, and cancer. SPOP forms linear higher-order oligomers following an isodesmic self-association model. Oligomerization is essential for SPOP's multivalent interactions with substrates, which facilitate phase separation and localization to biomolecular condensates. Structural characterization of SPOP in its oligomeric state and in solution is, however, challenging due to the inherent conformational and compositional heterogeneity of the oligomeric species. Here, we develop an approach to simultaneously and self-consistently characterize the conformational ensemble and the distribution of oligomeric states of SPOP by combining small-angle X-ray scattering (SAXS) and molecular dynamics simulations. We build initial conformational ensembles of SPOP oligomers using coarse-grained molecular dynamics simulations, and use a Bayesian/maximum entropy approach to refine the ensembles, along with the distribution of oligomeric states, against a concentration series of SAXS experiments. Our results suggest that SPOP oligomers behave as rigid, helical structures in solution, and that a flexible linker region allows SPOP's substrate binding domains to extend away from the core of the oligomers. Additionally, our results are in good agreement with previous characterization of the isodesmic self-association of SPOP. In the future, the approach presented here can be extended to other systems to simultaneously characterize structural heterogeneity and self-assembly.

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